This invention relates to methods for affecting viability of eucaryotic cells.
Normal human somatic cells (e.g., fibroblasts, endothelial, and epithelial cells) display a finite replicative capacity of 50-100 population doubling characterized by a cessation of proliferation in spite of the presence of abundant growth factors. This cessation of replication in vitro is variously referred to as cellular senescence or cellular aging, See, Goldstein, 249 Science 1129, 1990; Hayflick and Moorehead, 25 Exp. Cell Res. 585, 1961; Hayflick, ibid., 37:614, 1985; Ohno, 11 Mech. Aging Dev. 179, 1979; Ham and McKeehan, (1979) "Media and Growth Requirements", W. B. Jacoby and I. M. Pastan (eds), in: Methods in Enzymology, Academic Press, N.Y., 58:44-93. The replicative life span of cells is inversely proportional to the in vivo age of the donor (Martin et al., 23 Lab. Invest. 86, 1979; Goldstein et al., 64 Proc. Natl. Acad. Sci. USA 155, 1969; and Schneider and Mitsui, ibid., 73:3584, 1976), therefore cellular senescence is suggested to play an important role in aging in vivo.
Cellular immortalization (the acquisition of unlimited replicative capacity) may be thought of as an abnormal escape from cellular senescence, Shay et al., 196 Exp. Cell Res. 33, 1991. Normal human somatic cells appear to be mortal, i.e., have finite replicative potential. In contrast, the germ line and malignant tumor cells are immortal (have indefinite proliferative potential). Human cells cultured in vitro appear to require the aid of transforming viral oncoproteins to become immortal and even then the frequency of immortalization is 10.sup.-6 to 10.sup.-7. Shay and Wright, 184 Exp. Cell Res. 109, 1989.
Shay et al., 27 Experimental Gerontology 477, 1992, and 196 Exp. Cell Res. 33, 1991 describe a two-stage model for human cell mortality to explain the ability of Simian Virus 40 T-antigen to immortalize human cells. The mortality stage 1 mechanism (M1) is the target of certain tumor virus proteins, and an independent mortality stage 2 mechanism (M2) produces crisis and prevents these tumor viruses from directly immortalizing human cells. The authors utilized T-antigen driven by a mouse mammary tumor virus promoter to cause reversible immortalization of cells. The Simian Virus 40 T-antigen is said to extend the replicative life span of human fibroblast by an additional 40-60%. The authors postulate that the M1 mechanism is overcome by T-antigen binding to various cellular proteins, or inducing new activities to repress the M1 mortality mechanism. The M2 mechanism then causes cessation of proliferation, even though the M1 mechanism is blocked. Immortality is achieved only when the M2 mortality mechanism is also disrupted.
Harley et al., 345 Nature 458, 1990, state that the amount and length of telomeric DNA in human fibroblasts decreases as a function of serial passage during aging in vitro, and possibly in vivo, but do not know whether this loss of DNA has a causal role in senescence. They also state:
"Tumour cells are also characterized by shortened telomeres and increased frequency of aneuploidy, including telomeric associations. If loss of telomeric DNA ultimately causes cell-cycle arrest in normal cells, the final steps in this process may be blocked in immortalized cells. Whereas normal cells with relatively long telomeres and a senescent phenotype may contain little or no telomerase activity, tumour cells with short telomeres may have significant telomerase activity. Telomerase may therefore be an effective target for anti-tumour drugs.
There are a number of possible mechanisms for loss of telomeric DNA during ageing, including incomplete replication, degradation of termini (specific or nonspecific), and unequal recombination coupled to selection of cells with shorter telomeres. Two features of our data are relevant to this question. First, the decrease in mean telomere length is about 50 bp per mean population doubling and, second, the distribution does not change substantially with growth state or cell arrest. These data are most easily explained by incomplete copying of the template strands at their 3' termini. But the absence of detailed information about the mode of replication or degree of recombination at telomeres means that none of these mechanisms can be ruled out. Further research is required to determine the mechanism of telomere shortening in human fibroblasts and its significance to cellular senescence." [Citations omitted.]
Hastie et al., 346 Nature 866, 1990, while discussing colon tumor cells, state that:
"[T]here is a reduction in the length of telomere repeat arrays relative to the normal colonic mucosa from the same patient.
Firm figures are not available, but it is likely that the tissues of a developed fetus result from 20-50 cell divisions, whereas several hundred or thousands of divisions have produced the colonic mucosa and blood cells of 60-year old individuals. Thus the degree of telomere reduction is more or less proportional to the number of cell divisions. It has been shown that the ends of Drosophila chromosomes without normal telomeres reduce in size by 4 base pairs (bp) per cell division and that the ends of yeast chromosomes reduce by a similar degree in a mutant presumed to lack teiomerase function. If we assume the same rate of reduction is occurring during somatic division in human tissues, then a reduction in TRA by 14 kb would mean that 3,500 ancestral cell divisions lead to the production of cells in the blood of a 60-year old individual; using estimates of sperm telomere length found elsewhere we obtain a value of 1,000-2,000. These values compare favourably with those postulated for mouse blood cells. Thus, we propose that telomerase is indeed lacking in somatic tissues. In this regard it is of interest to note that in maize, broken chromosomes are only healed in sporophytic (zygotic) tissues and not in endosperm (terminally differentiated), suggesting that telomerase activity is lacking in the differentiated tissues." [Citations omitted.]
The authors propose that in some tumors telomerase is reactivated, as proposed for HeLa cells in culture, which are known to contain telomerase activity. But, they state:
"One alternative explanation for our observations is that in tumours the cells with shorter telomeres have a growth advantage over those with larger telomeres, a situation described for vegetative cells of tetrahymena." [Citations omitted.]
Harley, 256 Mutation Research 271, 1991, discusses observations allegedly showing that telomeres of human somatic cells act as a mitotic clock shortening with age both in vitro and in vivo in a replication dependent manner. He states:
"Telomerase activation may be a late, obligate event in immortalization since many transformed cells and tumour tissues have critically short telomeres. Thus, telomere length and telomerase activity appear to be markers of the replicative history and proliferative potential of cells; the intriguing possibility remains that telomere loss is a genetic time bomb and hence causally involved in cell senescence and immortalization.
Despite apparently stable telomere length in various tumour tissues or transformed cell lines, this length was usually found to be shorter than those of the tissue of origin. These data suggest that telomerase becomes activated as a late event in cell transformation, and that cells could be viable (albeit genetically unstable) with short telomeres stably maintained by telomerase. If telomerase was constitutively present in a small fraction of normal cells, and these were the ones which survived crisis or became transformed, we would expect to find a greater frequency of transformed cells with long telomeres." [Citations omitted.]
He proposes a hypothesis for human cell aging and transformation as "[a] semi-quantitative model in which telomeres and telomerase play a causal role in cell senescence and cancer" and proposes a model for this hypothesis.
De Lange et al., 10 Molecular and Cellular Biology 518, 1990, generally discuss the structure of human chromosome ends or telomeres. They state:
"we do not know whether telomere reduction is strictly coupled to cellular proliferation. If the diminution results from incomplete replication of the telomere, such a coupling would be expected; however, other mechanisms, such as exonucleolytic degradation, may operate independent of cell division. In any event, it is clear that the maintenance of telomeres is impaired in somatic cells. An obvious candidate activity that may be reduced or lacking is telomerase. A human telomerase activity that can add TTAGGG repeats to G-rich primers has recently been identified (G. Morin, personal communication). Interestingly, the activity was demonstrated in extracts of HeLa cells, which we found to have exceptionally long telomeres. Other cell types have not been tested yet, but such experiments could now establish whether telomerase activity is (in part) responsible for the dynamics of human chromosome ends."
Starling et al., 18 Nucleic Acids Research 6881, 1990, indicate that mice have large telomeres and discusses this length in relationship to human telomeres. They state:
"Recently it has been shown that there is reduction in TRA length with passage number of human fibroblasts in vitro and that cells in a senescent population may lack telomeres at some ends altogether. Thus in vitro, telomere loss may play a role in senescence, a scenario for which there is evidence in S. cerevisae and Tetrahymena. Some of the mice we have been studying are old in mouse terms, one and a half years,
yet they still have TRA's greater than 30 kb in all tissues studied. In humans, telomeres shorten with age at a rate of 100 bp per year, hence, it is conceivable that the same is happening in the mouse, but the removal of a few 100 bps of terminal DNA during its lifetime would not be detectable." [Citations omitted.]
D'Mello and Jazwinski, 173 J. Bacteriology 6709, 1991, state:
"We propose that during the life span of an organism, telomere shortening does not play a role in the normal aging process. However, mutations or epigenetic changes that affect the activity of the telomerase, like any other genetic change, might affect the life span of the individual in which they occur.
In summary, the telomere shortening with age observed in human diploid fibroblasts may not be a universal phenomenon. Further studies are required to examine telomere length and telomerase activity not only in different cell types as they age but also in the same cell type in different organisms with differing life spans. This would indicate whether telomere shortening plays a causal role in the senescence of a particular cell type or organism."
Hiyama et al., 83 Jpn. J. Cancer Res. 159, 1992, provide findings that "suggest that the reduction of telomeric repeats is related to the proliferative activity of neuroblastoma cells and seems to be a useful indicator of the aggressiveness of neuroblastoma . . . Although we do not know the mechanism of the reduction and the elongation of telomeric repeats in neuroblastoma, we can at least say that the length of telomeric repeats may be related to the progression and/or regression of neuroblastoma."
Counter et al., 11 EMBO J. 1921, 1992, state "loss of telomeric DNA during cell proliferation may play a role in ageing and cancer." They propose that the expression of telomerase is one of the events required for a cell to acquire immortality and note that:
This model may have direct relevance to tumourigenesis in vivo. For example, the finite lifespan of partially transformed (pre-immortal) cells which lack telomerase might explain the frequent regression of tumours after limited growth in vivo. In bypassing the checkpoint representing normal replicative senescence, transformation may confer an additional 20-40 population doubling during which an additional .apprxeq.2 kbp of telomeric DNA is lost. Since 20-40 doubling (10.sup.6 -10.sup.12 cells in a clonal population) potentially represents a wide range of tumour sizes, it is possible that many benign tumours may lack telomerase and naturally regress when telomeres become critically shortened. We predict that more aggressive, perhaps metastatic tumours would contain immortal cells which express telomerase. To test this hypothesis, we are currently attempting to detect telomerase in a variety of tumour tissues and to correlate activity with proliferative potential. Anti-telomerase drugs or mechanisms to repress telomerase expression could be effective agents against tumours which depend upon the enzyme for maintenance of telomeres and continued cell growth.
Levy et al., 225 J. Mol. Biol. 951, 1992, state that:
"Although it has not been proven that telomere loss contributes to senescence of multicellular organisms, several lines of evidence suggest a causal relationship may exist.
It is also possible that telomere loss with age is significant in humans, but not in mice." [Citations omitted.]
Windle and McGuire, 33 Proceedings of the American Association for Cancer Research 594, 1992, discuss the role of telomeres and state that:
"These and other telomere studies point in a new direction regarding therapeutic targets and strategies to combat cancer. If the cell can heal broken chromosomes preventing genomic disaster, then there may be a way to facilitate or artificially create this process. This could even provide a preventive means of stopping cancer which could be particularly applicable in high risk patients. The difference in telomere length in normal versus tumor cells also suggests a strategy where the loss of telomeres is accelerated. Those cells with the shortest telomeres, such as those of tumor metastasis would be the most susceptible."
Greider, 12 BioEssays 363, 1990, provides a review of the relationship between telomeres, telomerase, and senescence. She indicates that telomerase contains an RNA component which provides a template for telomere repeat synthesis. She notes that an oligonucleotide "which is complementary to the RNA up to and including the CAACCCCAA sequence, competes with d(TTGGGG)n primers and inhibits telomerase in vitro" (citing Greider and Blackburn, 337 Nature 331, 1989). She also describes experiments which she believes "provide direct evidence that telomerase is involved in telomere synthesis in vivo."
Telomeric DNA is usually composed of a simple repetitive sequence, with the strand running 5' to 3' from the end towards the center of the chromosome being C and A rich. For example, the telomeric sequence of the yeast S. cerevisiae is C.sub.1-3 A/TG.sub.1-3, and that of the ciliate Oxytricha is C.sub.4 A.sub.4 /T.sub.4 G.sub.4. The G-strand is extended to form a 12 to 16 base tail on some or all of the subchromosomal macronuclear DNA molecules in some ciliates. Single-strand TG.sub.1-3 tails .gtoreq.30 bases are present on yeast telomeres at the end of S-phase, and these tails support telomere-telomere interactions, at least in vitro (Wellinger et al., Cell, 72:51, 1993). In addition to the telomeric C.sub.1-3 A/TG.sub.1-3 sequences, many yeast chromosomes also have sub-telomeric middle repetitive elements called X and Y' that are interspersed with 80-130 bp of C.sub.1-3 A/TG.sub.1-3.
Since replication of linear chromosomes by conventional DNA polymerases would result in the progressive loss of DNA from their ends, telomeres are thought to require a specialized mechanism of replication (Watson, Nature 239:197, 1972). In several ciliates, and in human and mouse cells, the telomere-specific reverse transcriptase, telomerase, has been detected that adds telomeric repeats onto the ends of G-strand single-stranded telomeric substrates. Although the Tetrahymens telomerase is highly processive in vitro, typically synthesizing about 500 bases (Greider, Mol. Cell Biol. 11:4572, 1991), its processivity appears to be much lower in vivo (Yu and Blackburn, Cell, 67:823, 1991).
All telomerases have an essential RNA component that serves as the template for the addition of telomeric repeats. The substrate requirements for telomere formation in yeast in vivo (Murray and Szostak, Nature 305:189, 1983; Pluta et al., Proc. Natl. Acad. Sci. 81:1475, 1984) are similar to those for telomerase activity in vitro (Greider and Blackburn, Cell 43:405, 1985). In yeast, non-reciprocal recombination resulting in a net increase in telomeric DNA has been detected between plasmid-born telomeric tracts, a process that might also contribute to telomere replication (Pluta and Zakian, Nature 337:429, 1989; Wang and Zakian, Nature 345: 456, 1990). To at least some extent, the cellular machinery that replicates telomeres must be conserved among organisms with different telomeric sequences. Telomeric DNA from several organisms, including Oxytricha, can serve as substrates in yeast for the addition of yeast telomeric repeats (Pluta et al., 1984, supra) and the Tetrahymena telomerase can utilize different telomeric sequences as substrates for elongation (Greider and Blackburn, Cell 51:887, 1987).
Telomeric regions have additional properties that distinguish them from other regions of the genome. Genes that are near yeast telomeres are subject to telomeric position effect repression, which reversibly eliminates constitutive transcription, but does not affect induced transcription (Gottschling et al., Cell, 63:751, 1990). Yeast telomeric DNA is organized into a non-nucleosomal chromatin structure called the telosome (Wright et al., Genes & Dev., 6:1987, 1992). Telomeres of different chromosomes can be found associated with each other, with the nuclear envelope (Klein et al., J. Cell Biol. 117:935, 1992), and with the nuclear scaffold (de Lange, EMBO J. 11:717, 1992).
A number of yeast mutations have been found that affect telomeres. The Rap1 protein (Rap1p) has been shown to bind to telomeric sequences in vitro and in vivo (Buchman et al., Mol. Cell Biol. 8:5086-99, 1988; Longtine et al., Curt. Genet. 16:225, 1989; Conrad et al., Cell, 63:739, 1990; Klein et al., 1992, supra; Wright et al., 1992, supra). Some mutations in the essential RAP1 gene cause telomere shortening, while mutations or high levels of expression of the non-DNA binding carboxyl terminus of Rap1p cause telomere lengthening (Conrad et al., 1990, supra; Lustig et al., Science 250:549, 1990; Sussel and Shore, Proc. Natl. Acad. Sci. 88:7749, 1991; Kyrion et al., Mol. Cell. Biol. 12:5159, 1992). Mutations in the gene encoding Rif1p, which interacts with the carboxyl terminus of Rap1p in vivo, also cause telomere lengthening (Hardy et al., Genes & Dev., 6:801-14, 1992). Some mutations in CDC17, the catalytic subunit of DNA polymerase I, cause progressive telomere lengthening (Carson and Hartwell, Cell, 42:249, 1985), whereas mutations in the EST1, TEL1 and TEL2 genes cause telomere shortening (Lustig and Petes, Proc. Natl. Acad. Sci. 83:1398, 1986; Lundblad and Szostak, Cell, 57:633-43, 1989).
Schulz and Zakian, FASEB, Jul. 5, 1992 (not prior art to the present invention) at a yeast chromosomal structural meeting, presented an abstract which indicated that the PIF-1 helicase may be necessary for maintaining proper telomere length and stability.